CN109566410B - Culture method for in vitro culture of virus-free seedlings of cassava axillary bud somatic embryos - Google Patents
Culture method for in vitro culture of virus-free seedlings of cassava axillary bud somatic embryos Download PDFInfo
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Abstract
The invention provides a method for culturing a detoxified seedling by in vitro culture of an axillary bud cell embryo of cassava, which comprises the following steps: pre-culturing: taking cassava tender stems infected with ACMV (American Ginseng and vegetable juice) and cutting stem segments with axillary buds or stem segments with axillary buds of non-detoxified complete-virus-infected test-tube plantlets after conventional surface disinfection, and inoculating the stem segments with axillary buds to an axillary bud expansion induction culture medium for dark culture for 3-7 days; and (3) inducing and culturing somatic embryos: stripping the expanded axillary buds after culture, inoculating the axillary buds to a somatic cell embryo induction culture medium, and carrying out dark culture for 25-35 days; and (3) mature culture of somatic embryos: inoculating the somatic embryo tissue block to a somatic embryo maturation culture medium for illumination culture for 25-35 days; plant regeneration culture: inoculating the mature somatic cell embryo tissue block to a plant regeneration culture medium for illumination culture for 28-40 days. The invention has the advantages of high detoxification efficiency and propagation coefficient, more explant materials, simple and easy operation, high speed, more regenerated plants, high seedling rooting rate and strong growth vigor.
Description
Technical Field
The invention relates to a method for culturing a detoxified seedling by in vitro culture of a cassava axillary bud somatic embryo.
Background
Cassava (Manihot esculenta cratenz) is a plant of the Euphorbiaceae family (Euphorbiaceae), has a chromosome number of 2n ═ 36, perennial shrubs, and is 1-5m high. Cassava is the fourth largest tropical crop next to rice, corn and sorghum, and the root tuber of the cassava contains rich starch, so that the cassava can be used as food, industrial raw materials or feed and is a main food source for 6 hundred million people in tropical and subtropical regions. Cassava originated in south america and was introduced in africa, asia and oceania in the 16-18 th century. Cassava is now widely distributed in tropical and partially subtropical regions of the world. Cassava was introduced into China from south-east Asia Huaqiao 1820 years ago, and the cultivation history of cassava is more than 180 years. At present, cassava producing areas in China comprise provinces such as Hainan, Guangdong, Guangxi, Fujian and Yunnan, etc., and places such as Sichuan, Guizhou, Hunan and Jiangxi are also planted, the existing cultivation area is more than 50 million hectares, and the yield of fresh potatoes is 1000 million tons. The main advantage of cassava is its high yield potential. It is a C3-C4 intermediate crop, has high photosynthesis efficiency under proper conditions, and can maintain high photosynthesis efficiency under drought and high temperature stress.
The cassava mosaic disease is one of the most important viruses of cassava, the cassava yield loss is usually more than 50%, and the serious cassava yield loss can exceed 80%, so that the development of the cassava industry is seriously threatened. For farmers with low income in tropical regions taking cassava as main food, the economic benefit of cassava planting is greatly influenced, the food sources of growers can be seriously threatened, and extremely serious consequences are caused. Cassava mosaic disease is caused by many different species of Geminiviridae (Geminiviridae), and 7 viruses have been identified so far, including African Cassava Mosaic Virus (ACMV), East African cassava mosaic virus (East African cas sava mosaic virus), East African cassava mosaic Kemailong virus (East African cas sava mosaic virus), East African cassava mosaic kenyavirus (East African cas sava mosaic Keya virus), East African cassava mosaic malanavian virus (East African cas sava mosaic virus), East African cassava mosaic mulberry virus (East African cassava South African mosaic virus), and South African cassava mosaic virus (South African cassava mosaic virus).
The cassava mosaic virus can be transmitted by grafting, sap inoculation, seed stems and insect vectors, the phenomenon that a chlorosis spot appears on a leaf blade is started, then the chlorosis spot gradually expands and is mixed with surrounding normal green tissues to form a typical flower leaf, and the death of the whole cassava plant can be caused when the typical flower leaf is formed, so that great economic loss is caused. Due to the long-term asexual propagation, the yield of the cassava is reduced, the quality is reduced, the seed nature is degraded, once the mother plant is infected with the cassava mosaic virus, the virus is infected in the cassava body and is accumulated by generations. The tissue culture seedlings produced by the existing method still carry viruses, and the large-area occurrence of the mosaic disease of cassava is very easy to cause loss which is difficult to estimate after the transplantation, and the popularization of the cassava is also seriously influenced.
At present, the stem tip growing point of a plant is generally selected as an explant for producing the virus-free seedling, and the virus-free seedling is obtained by increasing the culture algebra. For example, the Chinese patent CN103202232A, based on the principle that the number of growing points of virus on the top of plant is far less than that of the mature part of plant, increases the number of generations of growing points to detoxify. However, the method for obtaining the virus-free seedlings needs to perform tissue culture by taking growth points for multiple generations, the culture period is long, the propagation efficiency of the virus-free seedlings is low, the large-scale popularization of the virus-free seedlings is influenced, and the introduction and the communication of high-quality cassava germplasm resources are also greatly hindered. In particular to one of the key factors for introduction of the virus-free seedlings by fast cultivation in the process of introducing cassava germ plasm resources from an epidemic area where the large-scale outbreak of the cassava mosaic virus occurs. In addition, the growing points of the stem tips are small in length and very young and tender, accurate sites are difficult to find quickly in the stripping process, the stems are very easy to break and damage in the stripping and transferring processes, the operation is very difficult, materials are wasted, and large-scale production is difficult.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides an efficient culture method for the cassava axillary bud somatic embryo in vitro culture virus-free seedlings.
The first aspect of the invention provides a culture method for culturing a virus-free seedling in vitro by a cassava axillary bud somatic embryo, which comprises the following steps:
and 3, mature culture of somatic embryos: inoculating the somatic embryo tissue block to a somatic embryo maturation culture medium for illumination culture for 25-35 days;
and 4, plant regeneration culture: inoculating the mature somatic embryo tissue block to plant regenerating culture medium for light culture for 28-40 days.
Wherein the axillary bud expansion induction medium is MS medium added with 5-20mg/L6-BA, and the addition amount of 6-BA can be, for example, 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, 10mg/L, 11mg/L, 12mg/L, 13mg/L, 14mg/L, 15mg/L, 16mg/L, 17mg/L, 18mg/L, 19mg/L or 20 mg/L. Preferably, the concentration of 6-BA in the axillary bud expansion induction culture medium is 10-20 mg/L.
Wherein the somatic cell embryo induction culture medium is added with 0.2-0.8mg/LCuSO in the MS culture medium4And 5-15mg/L of picloram. CuSO4The amount of (B) may be, for example, 0.2mg/L, 0.25mg/L, 0.3mg/L, 0.35mg/L, 0.4mg/L, 0.45mg/L, 0.5mg/L, 0.55mg/L, 0.6mg/L, 0.65mg/L, 0.7mg/L, 0.75mg/L, or 0.8 mg/L. The amount of picloram added may be, for example, 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, 10mg/L, 11mg/L, 12mg/L, 13mg/L, 14mg/L, or 15 mg/L. Preferably, the concentration of aminopyralid in the somatic embryo induction medium is 10-15 mg/L.
Wherein the somatic embryo maturation culture medium is MS culture medium added with 0.2-0.8mg/LCuSO40-0.1mg/L6-BA and 15-25g/L sucrose. CuSO4The amount of (B) may be, for example, 0.2mg/L, 0.25mg/L, 0.3mg/L, 0.35mg/L, 0.4mg/L, 0.45mg/L, 0.5mg/L, 0.55mg/L, 0.6mg/L, 0.65mg/L, 0.7mg/L, 0.75mg/L, or 0.8 mg/L. The amount of sucrose to be added may be, for example, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L, 20g/L, 21g/L, 22g/L, 23g/L, 24g/L, 25g/L, or the like. The amount of 6-BA added may be, for example, 0, 0.01mg/L, 0.02mg/L, 0.03mg/L, 0.04mg/L, 0.05mg/L, 0.06mg/L, 0.07mg/L, 0.08 mg/L, 0.09mg/L or 0.1 mg/L. Preferably, the concentration of 6-BA in the somatic embryo maturation medium is 0.01-0.05 mg/L.
Wherein the plant regeneration culture medium is prepared by adding 0.005-0.02mg/L6-BA, 0.01-0.03mg/L LGA3, 0.01-0.03mg/L LNAA and 15-20g/L sucrose into an MS culture medium. The amount of sucrose to be added may be, for example, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L, 20g/L, 21g/L, 22g/L, 23g/L, 24g/L, 25g/L, or the like. The amount of 6-BA added may be, for example, 0.005mg/L, 0.007mg/L, 0.008mg/L, 0.01mg/L, 0.012mg/L, 0.014mg/L, 0.015mg/L, 0.016mg/L, 0.018mg/L or 0.02 mg/L. The amount of GA3 added may be, for example, 0.01mg/L, 0.012mg/L, 0.014mg/L, 0.015mg/L, 0.016mg/L, 0.018mg/L, 0.02mg/L, 0.022mg/L, 0.024mg/L, 0.025mg/L, 0.026mg/L, 0.028mg/L, or 0.03 mg/L. The amount of NAA added may be, for example, 0.01mg/L, 0.012mg/L, 0.014mg/L, 0.015mg/L, 0.016mg/L, 0.018mg/L, 0.02mg/L, 0.022mg/L, 0.024mg/L, 0.025mg/L, 0.026mg/L, 0.028mg/L, or 0.03 mg/L.
Preferably, the plant regeneration culture medium is added with 6-BA at a concentration of 0.01mg/L, GA3 at a concentration of 0.02mg/L and NAA at a concentration of 0.02 mg/L.
The second aspect of the invention provides a culture medium group for culturing the cassava axillary bud somatic embryo in-vitro culture detoxified seedlings, which comprises an axillary bud expansion induction culture medium, a somatic embryo maturation culture medium and a plant regeneration culture medium.
Wherein the axillary bud expansion induction medium is MS medium added with 5-20mg/L6-BA, and the addition amount of 6-BA can be, for example, 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, 10mg/L, 11mg/L, 12mg/L, 13mg/L, 14mg/L, 15mg/L, 16mg/L, 17mg/L, 18mg/L, 19mg/L or 20 mg/L. Preferably, the concentration of 6-BA in the axillary bud expansion induction culture medium is 10-20 mg/L.
Wherein the somatic cell embryo induction culture medium is prepared by adding 0.2-0.8mg/LCuSO into MS culture medium4And 5-15mg/L of picloram. CuSO4The amount of (B) may be, for example, 0.2mg/L, 0.25mg/L, 0.3mg/L, 0.35mg/L, 0.4mg/L, 0.45mg/L, 0.5mg/L, 0.55mg/L, 0.6mg/L, 0.65mg/L, 0.7mg/L, 0.75mg/L, or 0.8 mg/L. The amount of picloram added may be, for example, 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, 10mg/L, 11mg/L, 12mg/L, 13mg/L, 14mg/L, or 15 mg/L. Preferably, the concentration of aminopyralid in the somatic embryo induction medium is 10-15 mg/L.
Wherein the somatic embryo maturation culture medium is MS culture medium added with 0.2-0.8mg/LCuSO40-0.1mg/L6-BA and 15-25g/L sucrose. CuSO4The amount of (B) may be, for example, 0.2mg/L, 0.25mg/L, 0.3mg/L, 0.35mg/L, 0.4mg/L, 0.45mg/L, 0.5mg/L, 0.55mg/L, 0.6mg/L, 0.65mg/L, 0.7mg/L, 0.75mg/L, or 0.8 mg/L. The amount of sucrose to be added may be, for example, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L, 20g/L, 21g/L, 22g/L, 23g/L, 24g/L, 25g/L, or the like. The amount of 6-BA added may be, for example, 0, 0.01mg/L, 0.02mg/L, 0.03mg/L, 0.04mg/L, 0.05mg/L, 0.06mg/L, 0.07mg/L, 0.08 mg/L, 0.09mg/L or 0.1 mg/L. Preferably, the concentration of 6-BA in the somatic embryo maturation medium is 0.01-0.05 mg/L.
Wherein the plant regeneration culture medium is prepared by adding 0.005-0.02mg/L6-BA, 0.01-0.03mg/L LGA3, 0.01-0.03mg/L LNAA and 15-20g/L sucrose into an MS culture medium. The amount of sucrose to be added may be, for example, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L, 20g/L, 21g/L, 22g/L, 23g/L, 24g/L, 25g/L, or the like. The amount of 6-BA added may be, for example, 0.005mg/L, 0.007mg/L, 0.008mg/L, 0.01mg/L, 0.012mg/L, 0.014mg/L, 0.015mg/L, 0.016mg/L, 0.018mg/L or 0.02 mg/L. The amount of GA3 added may be, for example, 0.01mg/L, 0.012mg/L, 0.014mg/L, 0.015mg/L, 0.016mg/L, 0.018mg/L, 0.02mg/L, 0.022mg/L, 0.024mg/L, 0.025mg/L, 0.026mg/L, 0.028mg/L, or 0.03 mg/L. The amount of NAA added may be, for example, 0.01mg/L, 0.012mg/L, 0.014mg/L, 0.015mg/L, 0.016mg/L, 0.018mg/L, 0.02mg/L, 0.022mg/L, 0.024mg/L, 0.025mg/L, 0.026mg/L, 0.028mg/L, or 0.03 mg/L.
Preferably, the plant regeneration culture medium is added with 6-BA at a concentration of 0.01mg/L, GA3 at a concentration of 0.02mg/L and NAA at a concentration of 0.02 mg/L.
The method provided by the invention has the advantages that the stem section containing the axillary buds is pretreated, the axillary buds are peeled after being expanded, and somatic embryo induction, somatic embryo maturation culture and mature somatic embryo plant regeneration culture are carried out to obtain the virus-free seedlings.
Drawings
FIG. 1 shows SC8 infested ACMV pot seedlings and SC5 and SC8 infected diseased and healthy leaves.
FIG. 2 is a photograph of the result of each stage of the in vitro detoxification culture of cassava axillary bud somatic embryos: A. expanding and culturing axillary buds; B. carrying out induction culture on the expanded axillary bud cell embryos; C. culturing somatic embryo; D. culturing a regeneration plant;
FIG. 3 shows the electrophoresis chart of RT-PCR detection of a part of regenerated plants: m.dl2000marker; 1-13, regenerating plants; 14. negative control; 15. a positive control;
FIG. 4 is an Actin electrophoretogram of the RT-PCR detection result of a part of regeneration plants: m.dl2000marker; 1-13, regenerating plants; 14. negative control; 15. and (4) positive control.
Detailed Description
The invention will be better understood from the following description of specific embodiments with reference to the accompanying drawings.
Preparation of ACMV cassava infection material
1. Material
The African Cassava Mosaic Virus (ACMV) DNA-A, DNA-B infectious clone is preserved in a potato biotechnology research laboratory of plant physiological and ecological research institute of Shanghai Life sciences of Chinese academy of sciences.
Agrobacterium tumefaciens (Agrobacterium tumefaciens): LBA4404
2. ACMV infection cassava by injection method and grafting method
The agrobacterium injection method comprises the following steps: and (3) transferring the cassava tissue culture seedlings into a greenhouse, planting the cassava tissue culture seedlings in an insect-proof net, wherein the temperature is 25 +/-2 ℃, the relative humidity is 60%, and the illumination/dark period is 16h/8 h. Agrobacterium LBA4404 containing ACMV-NOg DNA-A and DNA-B was streaked on YEB solid medium (Rif 25mg/L, Strep 100mg/L, Kan 50mg/L) and cultured at 28 ℃ for 48 hours. Then mixing the bacteria A and B, and repeatedly pricking the apical meristem of 5-6 leaf-stage cassava plants by using a sterile syringe with a needle.
Grafting method: adopting a cutting and grafting method, wherein the stock is an uninfected cutting potted seedling; the scions are tender stems infected with ACMV, and are cut into scions with 3-4 axillary buds and the length of about 8 cm. And cutting two sides of the lower end of the 3 cm-long position of the axillary bud of the scion into wedge-shaped inclined planes with the length of 2-3 cm. When the rootstock is thicker than the scion, the lower end of the scion is cut into a partial wedge shape, so that one side with the terminal bud is thicker, and the other side is slightly thinner, which is beneficial to joint sealing. When the thickness of the stock is consistent with that of the scion, the scion can be cut into a regular wedge shape, so that the stock can contain the clamp, and the contact surface of the stock and the scion is large, thereby being beneficial to healing. The surface of the scion needs to be flat and smooth, so that the cut surface is easy to abut against the cut of the stock, the cambium and the vascular bundle between the stock and the scion are tightly combined, and the cambium on the two surfaces is easy to heal.
3. Results of the experiment
When ACMV was successfully infected into SC5 and SC8 cassava by agrobacterium injection or grafting, the phenotype of the infection was seen: the leaves were chlorosis yellow and smaller than the control, the leaves were twisted (FIG. 1B, C), and then the virus phenotype appeared on both newly emerged leaves as systemic infection symptoms (FIG. 1A).
II, axillary bud somatic embryo in vitro detoxification culture
1. Material
The stem segment with axillary buds of SC8 potted plant infected with ACMV (African cassava mosaic virus) in artificial climate chamber of plant biotechnology research institute of Shanghai Life sciences academy of sciences of China, and the preparation method of SC8 potted plant is shown as 'preparation of ACMV cassava infection material'.
2. Screening for different 6-BA concentration pre-culture induced axillary bud expansion
Selecting tender stems of SC8 potted plants infected with ACMV, cutting stem segments with axillary buds on an ultraclean workbench after conventional disinfection treatment, inoculating the stem segments to MS basic culture media containing 6-BA at different concentrations of 5, 10, 15 and 20mg/L for axillary bud expansion induction and dark culture, inoculating 10 explants per treatment, repeating for 3 times, and observing the axillary bud expansion and growth conditions after 3d, 5d and 7 d.
After the stem segments with the axillary buds are inoculated for 3 days, the axillary buds can begin to sprout when the concentration of 6-BA is 10 and 20 mg/L. After 5d, the axillary buds treated by the treatment of 6-BA with the concentration of 10 and 20mg/L begin to expand, the expansion is particularly obvious after the treatment of 20mg/L, and the axillary buds are peeled off (figure 2, A); whereas the axillary buds treated with 5mg/L of 6-BA were seen to begin sprouting. After 7 days, the axillary buds treated with 6-BA with the concentration of 10 and 20mg/L are obviously expanded, and the base parts of internodes emerge bulges (such as the size of millet grains) with the diameter of 1-1.5mm, so the germination is very obvious; the axillary buds treated with the concentration of 6-BA of 5mg/L have no obvious expansion, and partial axillary buds grow leaves (not suitable for stripping). Considering the time saving, the axillary bud peeling can be carried out when the axillary bud sprouts and expands after the pre-culture is carried out for 5 days when the concentration of 6-BA is 20mg/L, and the optimal concentration for inducing the axillary bud to expand is obtained.
3. Screening of axillary bud callus and somatic embryo induced by different Picloram concentrations
Peeling the pre-cultured germinated and expanded axillary buds under a dissecting mirror, and inoculating the axillary buds to a tissue culture medium prepared from MS + CuSO40.5mg/L of basal medium, respectively adding 5, 10, 12 and 15mg/L Picloram (Picloram) somatic cell embryo induction medium to perform somatic cell embryo induction culture, inoculating 20 explants for each treatment, repeating for 3 times, and counting the callus induction rate and the somatic cell embryo induction rate after 30 days.
Callus induction rate (%) × (number of callus induced/number of explant inoculated) × 100%.
The somatic embryo induction rate (%) × (number of induced somatic embryos/number of inoculated explants) × 100%.
The callus induction rate difference of the Picloram with different concentrations on the expanded axillary buds is not obvious, and the somatic embryo induction rate difference is obvious (Table 1). When the concentration of Picloram is 10 and 15mg/L, the induction rate of somatic embryos is 83.3 percent, and no difference exists. Experiments show that when Picloram is 15mg/L, the callus grows excessively vigorously, and some somatic embryos are wrapped by the callus, so that the later growth of the somatic embryos is not facilitated; while at lower concentrations of Picloram, callus and somatic embryos germinated and grew slowly. Thus, the optimal expanded axillary bud cell embryo induced Picloram at 10 mg/L.
Comparing the concentrations of Picloram induced by the somatic embryos of the shoot tips and the expanded axillary buds of different explants, the optimal concentrations of the two are consistent. Comparing the induction rates of the callus and somatic embryo of the two explants, it can be seen that the induction rate of the somatic embryo of the expanded axillary bud is significantly higher than that of the micro-stem tip explant (fig. 2, B).
TABLE 1 Effect of different concentrations of Picloram on axillary bud callus and somatic embryo Induction
Description of the drawings: the significance test of Duncan's new range method shows that different lower case letters in the same column indicate significant difference between means (P < 0.05).
4. Screening of somatic embryo maturation culture with different 6-BA concentrations
Inoculating the obtained somatic embryo tissue block on a somatic embryo maturation culture medium: by MS + CuSO40.5mg/L + sucrose 20g/L is basal medium, 0, 0.01, 0.05, 0.1mg/L6-BA is added respectively for culture, illumination culture is carried out, 10 individual embryo blocks are inoculated in each treatment, and the process is repeated for 3 times; after 30 days of culture, counting the yield of somatic embryos and observing the growth condition.
Somatic embryo yield (one) is the total number of somatic embryos per number of explants from which somatic embryos were generated.
After somatic embryo tissue blocks were cultured in somatic embryo maturation medium for 30 days, statistical analysis of the mature cotyledon embryos of each tissue block revealed that the difference in the average somatic embryo yield between different 6-BA concentrations was not significant (Table 2). When the concentration of 6-BA is 0.01mg/L and 0.05mg/L, the average yield of somatic embryos reaches the highest, but the callus at the base of the somatic embryo tissue block grows more vigorously, which is not beneficial to nutrient absorption of the somatic embryos; moreover, the cotyledons of the culture medium contacting part are abnormally enlarged, which brings trouble to the subsequent transfer operation. A BA concentration of 0.01mg/L was found to be a suitable concentration for somatic embryo maturation in this study, considering growth and economy (FIG. 2, C).
TABLE 2 Effect of different 6-BA concentrations on somatic embryo maturation culture
Description of the drawings: the significance test of the Duncan's new complex pole difference method shows that different lower case letters in the same column indicate significant difference between mean numbers (P < 0.05)
5. Plant regeneration
Inoculating mature somatic embryos to plant regeneration medium: MS + BA 0.01mg/L + GA3Illumination culture is carried out on 0.02mg/L + NAA 0.02mg/L +20g/L sucrose, and the plant regeneration rate of the somatic embryo is counted after 35 d:
the average plant regeneration coefficient is the number of regenerated plants/total number of somatic embryo tissue blocks.
The mature somatic embryos are cultured on the regeneration plant culture medium for 35 days to obtain regeneration plants (figure 2, D), the average plant regeneration coefficient of each somatic tissue block is more than 3, the rooting rate of the regeneration plants is more than 95.0%, and the growth vigor is good.
6. Virus detection by RT-PCR
The leaves of healthy SC8 plants are used as negative control materials, the leaves of infected ACMVSC8 plants are used as positive control materials, and test-tube plantlets of somatic cell regeneration plants are used as detection materials.
Taking 0.1g of each of young leaves of a potted seedling and a test-tube seedling of an SC8 healthy and diseased plant (control), grinding by liquid nitrogen, respectively extracting total RNA, carrying out reverse transcription to form cDNA, carrying out amplification by adopting a 20-mu-L PCR reaction system, and carrying out analysis by 1% agarose gel electrophoresis. Designing primers according to the sequences of two main ACMV viruses, carrying out RT-PCR detection, and calculating the virus removal rate. The method comprises the following specific steps:
virus removal rate (%). plant number with virus removed/total plant number × 100
(1) PCR primer design
Comparing according to coding sequences of ACMV CP genes registered by GenBank, designing primers in conserved regions of the ACMV CP genes, wherein an upstream primer is named as AC1F, a downstream primer is named as AC1R, and an amplified fragment is about 240 bp; the primers for amplifying the reference gene in the semi-quantitative RT-PCR are designed according to an actin (beta-actin) gene 1, wherein an upstream primer is named as ActinF, a downstream primer is named as ActinR, and an amplification product is about 220 bp. The primers were synthesized by Biotechnology engineering (Shanghai) Ltd, and the sequences of the primers were as follows:
AC1F:5c-GGATTTTCCAGGAGCCACCA-3c；
AC1R:5c-GGCCCATATCGTCTTCCCTG-3c；
ActinF:5c-TGATGAGTCTGGTCCATCCA-3c；
ActinR:5c-CCTCCTACGACCCAATCTCA-3c
(2) extraction of total RNA from cassava
And (3) carrying out total RNA extraction on test materials of healthy cassava plants, ACMV-carrying diseased plants and virus-free test-tube plantlets. Total RNA extraction reference plant Total RNA extraction reagent (cat # DP437, Tiangen, Beijing, China) instructions.
The method mainly comprises the following steps: the tissue is rapidly ground in liquid nitrogen, then lysate is added to the ground tissue and fully shaken, the ground tissue is centrifuged at 12,000rpm at 4 ℃ for 10min, and the supernatant is transferred to a new RNase-free centrifuge tube. Add 0.1mL of 5M NaCl and mix gently. 0.3mL of chloroform was added thereto, and the mixture was inverted upside down and mixed. After the precipitate was filtered off, total RNA was collected by adsorption, centrifuged at 12,000rpm at 4 ℃ for 10min, and the upper aqueous phase was transferred to a new RNase-free centrifuge tube. Adding isopropanol with the same volume as the water phase, mixing, and standing at room temperature for 10 min. Centrifuge at 12,000rpm for 10min at 4 ℃. The supernatant was discarded and 1mL of 75% ethanol was added. Centrifuge at 12,000rpm for 5min at 4 ℃. Pouring out the liquid, and airing for 2-3min at room temperature. Adding 30-50 μ L RNase-free water, repeatedly beating and mixing, and dissolving RNA completely. Adding DNase I at 37 deg.C for 30min to eliminate genomic DNA contamination, then removing DNase I at 65 deg.C for 10min, and keeping at-70 deg.C for use. Taking 2uL total RNA to determine the optical density at 260 nm and 280nm, calculating the value of D260/D280, estimating the purity of the total RNA, and quantifying the total RNA of cassava through the value of D260. Total RNA was separated on a 1.0% normal agarose gel using 5uL of total RNA and the integrity of the total RNA was examined.
(3) Synthesis of cDNA 1 st Strand
Total mRNA was isolated from cassava leaves using the RNAprep Pure Plant kit (Tiangen, Beijing, China). cDNA was synthesized using reverse transcriptase, i.e., 2. mu.g of total RNA was synthesized in a 20. mu.L reaction system by reverse transcriptase ReverTra Ace (Code: TRT-101, TOYOBO, Shanghai). The 20 μ L standard reverse transcription system is specified below: mu.g of total RNA, 2.5. mu.L of 5pmoles Oligo (dT), 4. mu.L of 5 XBuffer, 2. mu.L of 10mM dNTPs, ReverTrace (100U/. mu.L), 20U of RNase Inhibitor, and 20. mu.L of DEPC water (or double distilled water without RNase) were added to make up.
(4) PCR amplification of ACMV specific primers and beta-actin gene
The cDNA synthesized as described above was used as a template, and the total reaction volume was 20. mu.L, which contained 1.0. mu.L of cDNA template, 2 XTAQPCR master mix 10.0. mu.L, and 1.0. mu.L of each of the upstream and downstream primers (or. beta. -actin gene), and the total volume was made up to 20.0. mu.L by adding ultrapure water.
The PCR reaction program is pre-denaturation at 95 ℃ for 5 min; the cycle conditions are denaturation at 95 ℃ for 40s, annealing at 58 ℃ for 50s, and extension at 72 ℃ for 60s, and 30 cycle numbers; extension at 72 ℃ for 10 min; keeping the temperature at 16 ℃.
The main reagents are as follows: plant total RNA extraction kit (Tiangen, Beijing);
PCR product recovery kit (Tiangen, Beijing);
real-time PCR detection kit (Toyobo, Shanghai);
the main apparatus is as follows: ND-1000 V3.7.0 ultraviolet spectrophotometer
Bio-Rad CFX96 fluorescent quantitative PCR instrument
The objective band of ACMV was stably detected from the positive control (ACMV-bearing diseased plant) by RT-PCR (fig. 4, lane 15), while the objective band of ACMV was not detected from the negative control (healthy plant) (fig. 4, lane 14). Of the 13 regenerated plant material tested in FIG. 1, 1 was amplified to bands, but bands were significantly lighter than the specific bands amplified by ACMV, i.e., less toxic (FIG. 4, lane 2); no target bands were amplified from the other 12 detoxified shoots (FIG. 4, lanes 1, 3-13). In 225 tested regenerated plant materials, the virus removal rate of 100 regenerated plants from the micro-stem tip somatic embryo and 5 detected target strips of the regenerated plants is 95.0 percent; the virus removal rate of 125 strains and 3 strains of axillary bud cell embryo regenerated plants detected target bands is 97.6 percent (figures 3 and 4).
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (4)
1. A method for culturing a virus-free seedling in vitro by culturing an axillary bud somatic embryo of cassava is characterized by comprising the following steps:
step 1, pre-culture: taking tender stems of cassava infected with ACMV, cutting stem segments with axillary buds or stem segments with axillary buds of non-virus-infected complete-virus test-tube plantlet after conventional surface disinfection, inoculating the tender stems to an axillary bud expansion induction culture medium, and carrying out dark culture for 3-7 days until the axillary buds expand, wherein the axillary bud expansion induction culture medium is an MS culture medium added with 10-20 mg/L6-BA;
step 2, inducing and culturing somatic cell embryos: stripping expanded axillary buds after culture, inoculating on somatic cell embryo induction culture medium added with 0.2-0.8mg/L CuSO in MS culture medium for dark culture for 25-35 days4And 10-15mg/L of picloram;
and 3, mature culture of somatic embryos: inoculating the somatic embryo tissue block to a somatic embryo maturation culture medium added with 0.2-0.8mg/L CuSO in MS culture medium for illumination culture for 25-35 days40.01-0.05 mg/L6-BA and 15-25g/L sucrose;
and 4, plant regeneration culture: inoculating the mature somatic embryo tissue block to a plant regeneration culture medium to perform illumination culture for 28-40 days, wherein the plant regeneration culture medium is prepared by adding 0.005-0.02mg/L6-BA, 0.01-0.03mg/L GA3, 0.01-0.03mg/L NAA and 15-20g/L cane sugar into an MS basal culture medium.
2. The culture method according to claim 1, wherein the concentration of 6-BA added to the plant regeneration medium is 0.01mg/L, the concentration of GA3 is 0.02mg/L, and the concentration of NAA is 0.02 mg/L.
3. A culture medium group for culturing the in vitro culture detoxified seedling of the somatic embryo of cassava axillary bud is characterized by comprising an axillary bud expansion induction culture medium, a somatic embryo maturation culture medium and a plant regeneration culture medium, wherein,
the axillary bud expansion induction culture medium is an MS culture medium added with 10-20 mg/L6-BA;
the somatic cell embryo induction culture medium is prepared by adding 0.2-0.8mg/L CuSO into MS culture medium4And 10-15mg/L of picloram;
the somatic embryo maturation culture medium is prepared by adding 0.2-0.8mg/L CuSO into MS culture medium40.01-0.05 mg/L6-BA and 15-25g/L sucrose;
the plant regeneration culture medium is prepared by adding 0.005-0.02mg/L6-BA, 0.01-0.03mg/L GA3, 0.01-0.03mg/L NAA and 15-20g/L sucrose into MS culture medium.
4. The culture medium group of claim 2, wherein the plant regeneration medium is supplemented with 6-BA at a concentration of 0.01mg/L, GA3 at a concentration of 0.02mg/L, and NAA at a concentration of 0.02 mg/L.
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